Deformation of the alveolar epithelial basement membrane with lung inflation has been implicated in blood-gas barrier breakdown during the development of ventilator-induced lung injury. To determine the vulnerability of alveolar epithelial cells to deformation-induced injury, we developed a cell-stretching device that subjects cells to cyclic, equibiaxial strains. Alveolar epithelial type II cells from primary culture were tested 1 and 5 days after seeding, during which time the cells underwent major morphological and phenotypic changes. Cells were subjected to changes in surface area of 12, 24, 37, and 50%, which corresponded to lung inflation of approximately 60, 80, 100, and >100% of total lung capacity. Deformation-induced injury of alveolar epithelial cells, assessed with a fluorescent cell viability assay, increased with deformation magnitude and decreased with time elapsed after seeding. In cells stretched after 1 day in culture, the percentage of dead cells after a single deformation ranged from 0.5 to 72% over the range of deformations used. In cells stretched at 5 days, the percentage of dead cells ranged from 0 to 9% when exposed to identical deformation protocols. These results suggest that morphological and phenotypic changes with time in culture fundamentally change the vulnerability of alveolar epithelial cells to deformation.